Parameters which distinguish phototaxis from random motility in Chlamydomonas reinhardtii have been defined with quantitative assays. The phototactic responses in photosynthetic, mixotrophic, and heterotrophic cultures were highest during exponential growth and declined rapidly as the cultures entered stationary phase. In contrast, random motility was relatively constant throughout growth. Phototaxis and motility also differ in their sensitivity to azide and antimycin A. Both of these drugs inhibited phototaxis within 5 min, but motility was unaffected for at least 30 min. Phototaxis and motility have different ion requirements. Optimum motility was observed in the presence of either Ca++ or Mg++; phototaxis required Ca++ and either K+ or NH4+. Photosynthesis is not required for phototaxis, since phototaxis was not inhibited by dichlorophenyldimethyl urea, and a mutant lacking chlorophyll was phototactic.

The effects of several organic and inorganic nitrogen compounds on nitrogenase mRNA and enzyme activity levels were examined in anaerobic cultures ofAnabaena variabiis 29413. Even low concentrations of exogenous ammonia (20 ,uM) Nitrogen-fixing microorganisms synthesize nitrogenase and the other proteins required for nitrogen fixation only under conditions in which nitrogen fixation is required to support growth. In the presence of ammonia and certain other fixed nitrogen compounds, the genetic capacity to fix nitrogen is not expressed. The effects of a variety of inorganic and organic nitrogen compounds such as amino acids and amino acid analogs on nitrogenase synthesis and activity have been studied in several nitrogen-fixing bacteria and cyanobacteria. Overall, the results of many studies show that other utilizable nitrogen sources are usually assimilated in preference to N2, and in the presence of many such compounds nitrogenase activity is absent or greatly reduced (1,10,27). In cyanobacteria, formation of heterocysts is also repressed by compounds which repress nitrogenase synthesis (10, 27). In most of the studies referred to above, investigators have measured nitrogenase activity (using the acetylene reduction technique), so that effects on nif gene regulation were not measured directly. In Klebsiella pneumoniae (3, 5) and Anabaena sp. strain 7120 (11), DNA-RNA hybridization techniques have been used to show that ammonia represses nif gene mRNA levels.As described in the preceding paper (12), we have defined conditions for studying derepression of nitrogenase genes of Anabaena variabilis under anaerobic conditions. This permits rapid experiments to be performed without the complication of heterocyst development. As we describe in this paper, we have used this anaerobic system to study the effects of various nitrogen compounds on nitrogenase gene expression at both transcription and enzyme activity levels. Our results support the conclusion that nitrogenous compounds influence nitrogenase levels primarily at the transcription level, probably by repressing transcription initiation, although some compounds may also have indirect effects on enzyme activity. Also, in agreement with most other studies that have not measured mRNA levels, we find that ammonia itself does not appear to be the immediate effector of nif gene expression. MATERIALS AND METHODSMedia and growth conditions. A. variabilis ATCC 29413 was used for all studies. Cultures were grown as described previously (14).Derepression experiments. Preparation of anaerobic cultures and incubation conditions for derepression experiments were as described previously (12). For experiments with NH4C1, KNO3, glutamine, and glutamate, a series of serum vial cultures was prepared containing cells in media (AA/8 with 0.5% fructose and 10 ,uM dichlorophenyldimethylurea) plus desired concentrations of the test compound; each experiment included a control vial containing no addition. The compound being tested was present throughout the experiment.Because carbamyl...

Gene clones encoding phycocyanin and allophycocyanin were isolated from an Anabaena variabilis ATCC 29413-Charon 30 library by using the phycocyanin (cpc) genes of Agmenellum quadruplicatum and the allophycocyanin (apc) genes of Cyanophora paradoxa as heterologous probes. The A. variabiis cpcA and cpcB genes occur together in the genome, as do the apcA and apcB genes; the two sets of genes are not closely linked, however. The cpc and apc genes appear to be present in only one copy per genome. DNA-RNA hybridization analysis showed that expression of the cpc and apc genes is greatly decreased during nitrogen starvation; within 1 h no cpc or apc mRNA could be detected. The source of nitrogen for growth did not influence expression of the genes; vegetative cells from nitrogen-fixing and ammonia-grown cultures had approximately the same levels of cpc and apc mRNAs. Heterocysts had less than 5% as much cpc mRNA as vegetative cells from nitrogen-fixing cultures. Northern hybridization (RNA blot) analysis showed that the cpc genes are transcribed to give an abundant 1.4-kilobase (kb) RNA as well as two less prominent 3.8-and 2.6-kb species. The apc genes gave rise to two transcripts, a 1.4-kb predominant RNA and a minor 1.75-kb form.

The human pathogen Eikenella corrodens expresses type IV pili and exhibits a phase variation involving the irreversible transition from piliated to nonpiliated variants. On solid medium, piliated variants form small (S-phase), corroding colonies whereas nonpiliated variants form large (L-phase), noncorroding colonies. We are studying pilus structure and function in the clinical isolate E. corrodens VA1. Earlier work defined the pilA locus which includes pilA1, pilA2, pilB, and hagA. Both pilA1 and pilA2 predict a type IV pilin, whereas pilB predicts a putative pilus assembly protein. The role of hagA has not been clearly established. That work also confirmed that pilA1 encodes the major pilus protein in this strain and showed that the phase variation involves a posttranslational event in pilus formation. In this study, the function of the individual genes comprising the pilA locus was examined using a recently developed protocol for targeted interposon mutagenesis of S-phase variant VA1-S1. Different pilA mutants were compared to S-phase and L-phase variants for several distinct aspects of phase variation and type IV pilus biosynthesis and function. S-phase cells were characterized by surface pili, competence for natural transformation, and twitching motility, whereas L-phase cells lacked these features. Inactivation of pilA1 yielded a mutant that was phenotypically indistinguishable from L-phase variants, showing that native biosynthesis of the type IV pilus in strain VA1 is dependent on expression of pilA1 and proper export and assembly of PilA1. Inactivation of pilA2 yielded a mutant that was phenotypically indistinguishable from S-phase variants, indicating that pilA2 is not essential for biosynthesis of functionally normal pili. A mutant inactivated for pilB was deficient for twitching motility, suggesting a role for PilB in this pilus-related phenomenon. Inactivation of hagA, which may encode a tellurite resistance protein, had no effect on pilus structure or function.Eikenella corrodens is a gram-negative bacterium native to the oral cavity and gastrointestinal tract in humans. This bacterium can also be pathogenic, causing a variety of soft tissue and wound infections (6, 9, 10, 16), endocarditis (4, 9), and other opportunistic infections. E. corrodens has also been associated with periodontal diseases (2,19,21), although a causal role has not been clearly established. Like several gram-negative pathogens including Neisseria gonorrhoeae, N. meningitidis, and Moraxella bovis, E. corrodens exhibits a phase variation that results from altered synthesis of type IV pili and is reflected in colony morphology changes. On solid medium, small (S-phase) corroding and large (L-phase) noncorroding colonies are observed (7,12,15,28). The L-phase variants arise irreversibly from S-phase variants at a frequency much greater than mutation rates. Colony morphology and phase variation correlates with the presence of pili on S-phase variants and the absence of pili on L-phase variants (11,12). Because type IV pili can be d...

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